New Light Microscope with Insane Resolution

Howard Hughes Medical Institute’s investigators have developed and tested a new type of optical microscope. Offering resolutions of only 2 to 25 nanometers, the new microscopy technology is essentially not limited by the wavelength of the visible light:

The prototype for the new microscope was assembled in September 2005 in the living room of one of the inventors, Harald Hess, who will soon become director of the applied physics and instrumentation group at Janelia Farm. Hess collaborated on the project with colleague Eric Betzig, who is now a group leader at Janelia Farm. The assembly and underlying conceptual work on the microscope were personally funded with $25,000 each from Hess and Betzig…
Hess and Betzig had both thought a lot already about how to build a better microscope. In 1993, Betzig published a paper in Science showing that the position of fluorescent molecules under a near-field optical microscope can be identified with precision far greater than the wavelength of light was once thought to allow. And in 1994, in work that was also published in Science, Hess and Betzig showed together that the closely packed points light in a semiconductor could be individually isolated and studied. Combining these earlier studies, Betzig later proposed that molecular-level resolution might be achieved in cells by imaging just a few molecules at a time and identifying each one’s center – but no one yet knew how to separate out a cell’s densely packed proteins to make that possible under physiological conditions.
It was a tool developed in a biology lab that ultimately inspired the two physicists plan to build a better microscope. “In the world of biology there is a new generation of fluorescent proteins that you can switch on at will with a little bit of violet light,” Hess explained. He and Betzig learned of these molecules, which biologists can genetically link to cellular proteins that they wish to study, during conversations with Florida State University (FSU) scientist Michael Davidson in April 2005…
The basic concepts behind their new technique are simple: The researchers label the molecules they want to study with a photoactivatable probe, and then expose those molecules to a small amount of violet light. The light activates fluorescence in a small percentage of molecules, and the microscope captures an image of those that are turned on until they bleach. The process is repeated approximately 10,000 times, with each repetition capturing the position of a different subset of molecules.
Because the number of molecules captured in each image is small, they are far enough apart to see each molecule individually and thereby localize its center, Hess said. When a final image is created that includes the center of each individual molecule, it has a resolution previously only achievable with an electron microscope. Unlike electron microscopy, however, the new technique allows for more flexibility in labeling molecules of interest.
By early 2006, the collaborators were taking strikingly clear images of a number of cellular structures, including actin filaments, focal adhesion proteins, mitochondria, and the Golgi apparatus. “Performance-wise, it’s just astounding. And simplicity-wise it’s astounding,” Betzig said. “It knocks the socks off of standard fluorescence microscopy from a resolution perspective…”

Picture caption by HHMI: Resolution limitations of traditional optical microscopes lead to blurry images of small cellular components such as mitochondria (level 1). However, if specific proteins are tagged with fluorescent molecules that can be activated one-at-a-time (between levels 1 and 2), the positions of the molecules can be determined at a much finer level. The total position data is then assembled into an image (level 2) at a resolution comparable to an electron microscope (level 4), but with the added benefit of being specific to only the desired target protein.LINK…

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